Project Summary Glycans play key roles in all aspects of biology. While harboring untapped potential as a target for biomedical research, the glycome is still poorly characterized with respect to composition and function. In the absence of a molecular template for glycan assembly, genetic, enzymatic and metabolic methods for the engineering of cell surface glycan displays have been instrumental in establishing our current understanding of the physiological and pathophysiological functions of glycans. While powerful, the current glycan engineering tools have not yet yielded full control over the composition and structure of glycans that can be installed on living cells. A significant challenge in glycan engineering is the delivery of the nucleotide sugar building blocks of glycans and various modulators of glycosylation (e.g., inhibitors of glycosylation enzymes) into the organelles, where the glycosylation machinery of cells is localized (i.e., the Endoplasmic Reticulum and the Golgi compartment). These chemical agents are often polar or charged, and are unable to cross the multiple cellular membranes separating the extracellular space from the secretory compartments. This proposal describes the development of a method for delivery of cell-impermeable nucleotide sugars and glycosylation inhibitors directly from the culture medium into the Golgi, bypassing the cytosolic compartment. The new method capitalizes on the intracellular trafficking of lipids between the plasma membrane and various cellular organelles. The proposal identifies lipids at the outer leaflet of the plasma membrane as potential carriers to shuttle cargo into the lumen of the Golgi compartment. The proposed work will establish 1) structure-activity relationships for lipid modifications that maximize the delivery of chemical cargo from the cell surface into the Golgi lumen, 2) optimal bioconjugation chemistries for the loading of nucleotide sugars and glycosyl transferase inhibitors and their release in the Golgi lumen environment, and 3) high-payload macromolecular scaffolds for the delivery and release of glycosylation modulators into the Golgi. A key feature of the proposed method will be the ability to alter the composition of cell surface glycans without relying on endogenous biosynthetic and salvage pathways for the generation of nucleotide sugars as well as transporters required for their translocation from the cytosol into the Golgi. Therefore, the proposed method will overcome current limits on glycan structures accessible using metabolic oligosaccharide engineering. It is also poised to offer a general mechanism for the treatment of congenital disorders of glycosylation caused by defects in nucleotide sugar biosynthesis and transport, an approach for addressing various pathophysiologies associated with aberrant glycosylation, and an improved method for tuning glycosylation profiles of biologics and tissue replacements produced in non-human organisms.